cytokine cocktail m5  (PeproTech)

Journal: Molecular Medicine

Article Title: Paris saponin VII attenuates psoriasiform inflammation by regulating STAT3/NFκB signaling pathway and Caspase-1-induced pyroptosis

doi: 10.1186/s10020-025-01253-y

Figure Lengend Snippet: PSVII exhibited anti-inflammatory and antioxidant effects in IMQ-induced psoriasis-like mice. ( A ) ELISA analysis of serum levels of IL-17, IL-23, IL-2, IL-6 and TNF-α. ( B ) and ( C ) Relative expression of TNF-α in PSVII -treated psoriasis models in vitro. ( D ) Immunofluorescence microscopy of HaCaT sections treated with PSVII , probing for TNF-α infiltration (scale bar = 10 μm). ( E ) ROS expression in various groups, as observed under fluorescence microscopy (scale bar = 20 μm). n = 6 mice/group, n = 3 cells/group. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. Model group

Article Snippet: The cells were divided into the following groups: (Griffiths et al. ) Control group; (Chandran ) Model group; and (Lee and Kim ) PSVII treatment groups, which received PSVII at concentrations of 2 μM, 5 μM, and 10 μM (designated as PSVII -L, PSVII -M, and PSVII -H, respectively), for 24 h. A psoriasis-like model was induced in the model group using M5 (The cytokine mixture composed of IL-17, IL-22, IL-1α, TNF-α, and Oncostatin M, the concentration was 10 ng/ml, Novoprotein, China), as previously described (Zhao et al. ).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, In Vitro, Immunofluorescence, Microscopy, Fluorescence

Journal: Frontiers in Immunology

Article Title: Identification and validation of shared biomarkers and drug repurposing in psoriasis and Crohn’s disease: integrating bioinformatics, machine learning, and experimental approaches

doi: 10.3389/fimmu.2025.1587705

Figure Lengend Snippet: Validation of hub genes via cellular experiments. (A) CCK-8 assay showing the proliferation capacity (OD value) of cells in the M5-treated group and the control group at different time points. (B-F) qPCR validation of the relative expression levels of hub genes in the psoriasis cell model treated with M5. (G-I) qPCR analysis of inflammatory cytokines IL-6, IL-8, and TNF-α expression levels in the CD cell model stimulated with LPS. (J-N) qPCR validation of the relative expression levels of hub genes in the CD cell model stimulated with LPS. Statistical significance: ns indicates no significance, * P < 0.05, ** P < 0.01, *** P < 0.001. Data are presented as mean ± SEM.

Article Snippet: M5 cytokines sourced from PeproTech (Rocky Hill, USA) were utilized in this study.

Techniques: Biomarker Discovery, CCK-8 Assay, Control, Expressing

Journal: Frontiers in Immunology

Article Title: Identification and validation of shared biomarkers and drug repurposing in psoriasis and Crohn’s disease: integrating bioinformatics, machine learning, and experimental approaches

doi: 10.3389/fimmu.2025.1587705

Figure Lengend Snippet: Effects of Etoposide on HaCaT and HT-29 cell lines. (A) Chemical structure of Etoposide. (B) CCK-8 assay showing the effect of different concentrations of Etoposide (1, 5, 10, 20, and 50 μM) on HaCaT cell viability. (C, D) qRT-PCR analysis of the expression levels of psoriasis-related marker genes KRT6 and KRT16 in HaCaT cells. HaCaT keratinocytes were treated with or without 1 μM Etoposide for 24 hours and simultaneously stimulated with M5 cocktail cytokines (10 ng/ml) or not for 24 hours. Cells were harvested, and RNA was extracted for qRT-PCR analysis, with β-actin as an internal reference. The following groups were included: DMSO (Control), DMSO+M5 (M5), M5+Etoposide (M5+Etoposide). (E) CCK-8 assay showing the effect of different concentrations of Etoposide (1, 5, 10, 20, and 50 μM) on HT-29 cell viability. (F-H) qRT-PCR analysis of the expression levels of CD-related inflammatory cytokines IL6, IL8, and TNF-α in HT-29 cells. HT-29 cells were treated with or without 1 μM Etoposide for 24 hours and simultaneously stimulated with LPS (20 μg/ml) or not for 24 hours. Cells were harvested, and RNA was extracted for qRT-PCR analysis, with β-actin as an internal reference. The following groups were included: DMSO (Control), DMSO+LPS (LPS), LPS+Etoposide (LPS+Etoposide). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: M5 cytokines sourced from PeproTech (Rocky Hill, USA) were utilized in this study.

Techniques: CCK-8 Assay, Quantitative RT-PCR, Expressing, Marker, Control

Journal: bioRxiv

Article Title: PEPITEM Tripeptides and Peptidomimetics: Next-Generation Modulators of Inflammation in Immune-Mediated Conditions

doi: 10.1101/2024.10.14.618151

Figure Lengend Snippet: The effect of PEPITEM and SVT[NH-Ethyl] is mirrored in the lymphoid organ repertoire. A) Spleen weight/mouse body weight ratio (expressed as g) evaluated at the experimental endpoint (day 7). Flow cytometry analysis of splenic B) CD4 + , C) CD8 + , D) Th1 (CD4 + IFN-γ + ) and E) Th17 (CD4 + IL-17 + ) subsets, pre-gated on living cells (see flow cytometry strategy reported in Supplementary Fig. 2B and C). Histograms indicate the total positive populations (expressed as x10 6 ) in the different experimental conditions. F) Quantification of T-related cytokines performed by a multi-LEGENDplex ™ analyte flow array (gating strategy is reported in Supplementary Fig. 3) on lymphocytes isolated from skin-draining lymph nodes, following ex vivo stimulation with PMA and ionomycin, presented as a heatmap (colormap:double gradient) and expressed as pg mL −1 . G), H) and I) TNF-α, IL-6 and IL-17A cytokines extrapolated from the heatmap and represented graphically as histograms. Data are presented as mean ± S.D. of n=6 animals in each group. Statistical analysis was conducted by one- or two-way ANOVA followed by Bonferroni’s or Dunnett’s posthoc-test. ## P ≤ 0.01, ### P ≤ 0.001, #### P ≤ 0.0001 vs CTRL group; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 vs Imiquimod group.

Article Snippet: Psoriasis-like keratinocytes model was established by stimulating HaCaT cells with M5 cytokines cocktail consisting of 2.5 ng mL −1 of each TNF-α (210-TA/CF), Oncostatin M (295-OM/CF), IL-1α (200_LA/CF), IL-17 (317-ILB) and IL-22 (782-IL/CF, all from R&D System, Italy) as previously described , .

Techniques: Flow Cytometry, Isolation, Ex Vivo

Journal: bioRxiv

Article Title: PEPITEM Tripeptides and Peptidomimetics: Next-Generation Modulators of Inflammation in Immune-Mediated Conditions

doi: 10.1101/2024.10.14.618151

Figure Lengend Snippet: A) and B) In vitro mechanistic studies on PEPITEM, tripeptides and peptidomimetics. In vitro cytotoxic examination, evaluated by MTT assay, for PEPITEM, tripeptides and peptidomimetics performed on J774A.1 murine macrophage cell lines, following 4 and 24 h of treatment with the selected concentrations (1-30 ng mL −1 ). Dotted lines indicate 75% of cell viability. Data are expressed as cell viability (% of control) and presented as mean ± S.D. of 3 independent experiments. C) and D) IL-6 and TNF-α ELISA assays performed on supernatant of J774A.1 pre-treated with indicated compounds at the concentration of 10 ng mL −1 (highest non-cytotoxic concentration) and then stimulated with LPS (10 µg mL −1 ) for 24 h. E) and F) IL-6 and TNF-α ELISA assays performed on supernatants of NIH-3T3 mouse fibroblasts pre-treated with indicated compounds at the concentration of 10 ng mL −1 and then stimulated with LPS (10 µg mL −1 ) for 24 h. ( C-F ) Data are expressed as pg mL −1 and presented as means ± S.D. of 3 independent experiments. Statistical analysis was conducted by one-way ANOVA followed by Bonferroni’s posthoc-test. # P ≤ 0.05, ### P ≤ 0.001, #### P ≤ 0.0001 vs CTRL group; *P ≤ 0.05, **P ≤ 0.01 vs LPS group. G) Effect of tested compounds on hyperproliferation of M5 cytokines (TNF-α, Oncostatin M, IL-1α, IL-17 and IL-22)-stimulated HaCaT cells, as in vitro psoriasis-like model. HaCaT hyperproliferation (expressed as cells proliferation, % of control) was measured using MTT assay and presented as mean ± S.D. of 3 independent experiments. Statistical analysis was conducted by one-way ANOVA followed by Bonferroni’s posthoc-test. *P ≤ 0.05 vs M5 group.

Article Snippet: Psoriasis-like keratinocytes model was established by stimulating HaCaT cells with M5 cytokines cocktail consisting of 2.5 ng mL −1 of each TNF-α (210-TA/CF), Oncostatin M (295-OM/CF), IL-1α (200_LA/CF), IL-17 (317-ILB) and IL-22 (782-IL/CF, all from R&D System, Italy) as previously described , .

Techniques: In Vitro, MTT Assay, Control, Enzyme-linked Immunosorbent Assay, Concentration Assay